Therapeutic uses of SMR1 peptides

ABSTRACT

A method for treating or reducing the severity of a disease or disorder mediated by a membrane metallopeptidase by administering to a mammal a SMR1-peptide or a pharmaceutically active amount of a SMR1-peptide.

In a first aspect, the invention relates to new therapeutic uses of aSMR1-peptide.

The inventors have previously characterized a new rat submandibulargland protein, named SMR1 (submandibular rat 1 protein), which has thestructure of a prohormone and whose synthesis is under androgen control(Rosinsky-Chupin et al., (1988), Proc. Natl. Acad. Sci. USA;85(22):8553–7) and PCT Patent Application No. WO 90/03981). The geneencoding SMR1 belongs to a new multigene family, the VCS family, whichhas been localized to rat chromosome 14, bands p21–p22 (Courty et al.,(1996) Mol. Biol. Evol. 13(6):758–66; Rosinsky-Chupin et al., (1995)Mamm. Genome 6(2): 153–4)) and for which some human gene members havebeen characterized (Isemura et al., (1997), J Biochem 121:1025–1030;Isemura et al. (1994) J Biochem 115:1101–1106; Isemura et al. (1979) JBiochem 86:79–86; Dickinson et al. (1996) Curr Eye Res 15:377–386). Thegene has an organization similar to a number of hormone precursor genes(Rosinsky-Chupin et al., (1990) DNA Cell. Biol. 9(8):553–9). SMR1 mRNAis expressed in a highly tissue-, age- and sex-specific manner in theacinar cells of the male rat submaxillary gland (SMG) and in theprostate (Rosinsky-Chupin et al., (1993) Histochem. Cytochem.41(11):1645–9).

It has been described that, in vivo, SMR1 is selectively processed atpairs of basic amino acid sites in a tissue- and sex-specific manner togive rise to mature peptide products, in a manner similar to thematuration pathway of peptide-hormone precursors (Rougeot et al., (1994)Eur. J. Biochem. 219(3):765–73). The structurally related peptidesgenerated from SMR1 by cleavage at pairs of arginine residues (e.g. theundecapeptide: VRGPRRQHNPR (SEQ ID NO: 4); the hexapeptide: RQHNPR (SEQID NO: 9); and the pentapeptide: QHNPR (SEQ ID NO: 2)) are in vivoselectively matured from the precursor after processing at pairs ofbasic amino acid residues by a paired basic amino acid-convertingenzyme, likely the Furine convertase,—differentially accumulated in atissue-, age- and sex-related manner, and—locally as well assystemically released upon multifactorial neuroendocrine control(Rougeot et al, 1994).

In such a context, the final mature peptide generated from SMR1, namedSMR1-Pentapeptide (SMR1-QHNPR (SEQ ID NO: 2)), also named sialorphin, issynthesized predominantly in response to androgen steroids and isconstitutively released into the bloodstream in basal condition andacutely released in response to environmental stress, depending on thestate of activation of adrenoreceptors controlling the secretoryresponsiveness of the SMG.

In turn, the circulating SMR1-Pentapeptide is in vivo rapidly andselectively taken up by peripheral targets through specific bindingsites, predominantly within renal, bone and dental tissues.

The fact that the target sites of the peptide are mainly localizedwithin the major tissues of ion capture, transport and regulation, givesevidence that SMR1-Pentapeptide might play a local and systemic role inmodulating mineral ion homeostatic process, in vivo. Furthermore,associated with the fact that the androgen-regulated SMR1-Pentapeptideis upon environmental stress acutely secreted, these findings led theinventors to postulate that this SMG-specific signaling peptide mightparticipate in mediating integrative reestablishment of dynamichomeostatic responses: to stressful situations within male rat-specificbehavioral characteristics such as aggressive and/or sexualintercourses, and in relation to female-specific physiologicalcharacteristics such as pregnancy and lactation.

WO 98/37 100 discloses that the maturation products of the SMR1 protein,specifically the peptide of structural formula XQHNPR (SEQ ID NO: 10),recognize specific target sites in organs that are deeply involved inthe mineral ion concentration. This discovery has led the inventors toassign to the SMR1-peptide (especially the SMR1-pentapeptide,hexapeptide or undecapeptide) an active role in the regulation of themetal ion concentrations in the body fluids and tissues, and thus atherapeutic role of these peptides in all the metabolic disordersrelated to a mineral ion imbalance.

Namely, the therapeutic peptides disclosed therein are useful fortreating or preventing bone, teeth, kidney, intestine, pancreas,stomach, or salivary gland disorders caused by a mineral ion imbalancein the body fluids or tissues, namely hyper- or hypo-parathyroidism,osteoporosis, pancreatitis, submandibular gland lithiasis,nephrolithiasis or osteodystrophy.

On the basis of the hypothesis mentioned above, a behavioralpharmacological approach has been undertaken. SMR1-peptide, especiallySMR1-Pentapeptide was found to induce a dose-dependent improvement onthe sexual behavior of adult male rats with a loss of the aggressiveimpulse behavior seen in control rats (PCT patent application WO 01/00221).

To elucidate the pathways that have taken place in the SMR1-peptideaction, one of the essential steps was to investigate the molecularcharacteristics of the peptide-receptor sites. The isolation of themembrane binding site accessible to the systemic administration orradiolabelled SMR1-Pentapeptide, especially within the renal outermedulla has been achieved. The identification of its amino-acid sequencehas revealed that the cell surface molecule which binds the peptide invivo, is a membrane metallopeptidase and more specifically a mammaliantype II integral membrane zinc-containing endopeptidase, i.e. NeutralEndoPeptidase 24-11 or NEP, also named Enkephalinase that belongs to theNeprilysin subfamily, which plays critical role in the functionalpotency of various peptidergic signals. Moreover, the in vivo directinteraction of rat kidney NEP and SMR1-Pentapeptide was demonstrated invitro using purified rabbit kidney NEP.

Furthermore, at the level of whole rat body a good (topological andkinetical) correspondence was found in vivo between the distribution oftarget organs accessible to circulating radiolabelled SMR1-Pentapeptideand that of known synthetic NEP inhibitor (3HHACBO-Gly) (Sales et al,(1991) Regulatory Peptides 33, 209–22). Otherwise, a number ofobservations argue for the hypothesis that SMR1-peptide is a SMG-derivednatural modulator, especially an inhibitor, of the NEP activity:

1—the SMR1-Pentapeptide tissue uptake was found to bepharmacokinetically and biochemically stable in vivo,

2—the SMR1-peptide does not share the residues required to be a NEPsubstrate, seeing that the NEP preferentially cleaves peptides betweenthe X-Phe bond, and

3—the SMR1-Pentapeptide has strong zinc-chelating group, which has beendesigned for the potent synthetic NEP inhibitors.

In view of the numerous physiological NEP substrates (namely the peptidehormones: Enkephalins, Substance P, Bradykinin, Angiotensin II andatrial natriuretic peptide), physiological consequences of theNEP/SRM1-peptide interaction are expected to impact on the control ofcentral and peripheral pain perception, inflammatory phenomena, arterialtone and/or mineral exchange (Roques et al, 1993 infra).

Neutral endopeptidase, NEP 24-11, is distributed both in nervous andperipheral tissues of mammals, and in the periphery it is particularlyabundant in the kidney and placenta. In these tissues the cell-surfacemetallopeptidase NEP participates in the postsecretory processing andmetabolism of neuropeptides, systemic immunoregulatory peptides andpeptide-hormones. By controlling the active levels of circulating orsecreted regulatory peptides, NEP modulates their physiologicalreceptor-mediated action. Hence, the membrane-anchored NEP is involvedin regulating the activity of: potent vasoactive peptides such asSubstance P, Bradykinin (BK), Atrial Natriuretic peptide (ANP), andAngiotensin II (AII); potent inflammatory/immunoregulatory peptides suchas Substance P and BK and fMet-Leu-Phe (fMLP); potent opioidneuropeptides such as Met and Leu-Enkephalins (Enk) and potent mineralexchange and fluid homeostasis regulatory peptides such as ANP, C-typeNatriuretic Peptide (CNP) and B-type Natriuretic Peptide (BNP). Howeverthe levels of these peptides are changed through the NEP-inducedformation/degradation only in regions where they are tonically releasedor where their release is triggered by a stimulus.

From an integrative point of view, the NEP biological activity is tocontrol the active levels of peptidergic signals involved in arterialtension regulation, in inflammatory phenomena and in water-mineralhomeostasis, as well as, in the control of pain processing. From aclinical point of view, this substantiates the fact that NEP is animportant drug target in various disease states. For example, byinhibiting NEP, thereby increasing the levels and duration of action ofcentral or peripheral endogenous opioids, an analgesic or antidiarrhealagent could be obtained, or by inhibiting endogenous AII formation andsubstance P, BK and ANP inactivation, antihypertensive, natriuretic anddiuretic agents could be obtained. The main advantage of modifying theconcentrations of endogenous peptides by use of NEP inhibitors is thatthe pharmacological effects are induced only at receptor stimulated bythe natural effectors, and are critically dependent on the tonic orstimulus-evoked release of the natural effectors happening uponenvironmental, behavioral and physiopathological stressful situations(Roques et al, (1993) Pharmacological Reviews 45, 87–146). It isimportant to stress that in such stressful context, the naturalpotential NEP-modulator, SMR1-peptide, will be also acutely andtonically released, distributed and taken up by its systemic targettissues, especially by the renal NEP sites (Rougeot et al, 1997).Thereby, the SMR1-peptide would be in vivo kinetically bioavailable tomodulate NEP activity and so to optimize the local and systemicinflammatory, pressor and/or ion homeostatic responses to stress. Theintegrative point of view is in concordance with the assumption thatcirculating Submaxillary Gland (SMG)-derived factors might participatein integrative reestablishment of homeostatic responses to physiologicalor pathological “stress states” (injury, trauma or infection), ratherthan contribute to the resting homeostatic steady state (Rougeot et al,(2000) Peptides 21, 443–55).

From a general point of view, evidence of a physiological significancedemonstrates the existence of a Cervical Sympathetic Trunk (CST)-SMGneuroendocrine axis that plays an integral role in physiologicaladaptations and contributes to the maintenance of homeostasis inmammals, especially under the “stress conditions” seen in rodents withtissue damage, inflammation, and aggressive behavior. The data gatheredin the laboratory provide convincing evidence that SMR1-peptide is anovel signaling mediator, adapted to the sex, and species-specificenvironmental, behavioral and physiological characteristics, tonicallyand dynamically mobilized upon urgent situations, in the way to optimizeboth local and systemic nociceptive, inflammatory, pressor and/or ionhomeostatic responses, through regulation of the membrane-bound NEPactivity. Otherwise, the SMR1-peptide, which is to date the firstnatural regulator of the peripheral NEP activity identified, seems to bedesigned as a new class of therapeutic molecules as thismetallopeptidase is well-conserved especially between rat, rabbit andhuman species with sequence homology ≧90%.

The evidence provided by the inventors together with the strikinghomology with the NEP sequences between species further suggest that theSMR1-peptide may act as natural modulator/inhibitor of membranemetallopeptidases, notably zinc metallopeptidases (GenBank Access numberP 08473, Malfroy et al, (1988) FEBS Lett. 229(1), 206–210; NP 258428,Bonvouloir et al, (2001) DNA Cell Biol. 20(8), 493–498; NP 036740,Malfroy et al. (1987) Biochem Biophys Res Commun 144, 59–66).

Examples of mammalian membrane metallopeptidases besides NEP are ECE(Endothelin-Converting Enzymes), in particular ECE1 and ECE2, theerythrocyte cell-surface antigen KELL and the product of PEX geneassociated with X-linked hypophosphatemic rickets, as well as ACE(Angiotensin Converting Enzyme) and APN (Aminopeptidase N).

Inhibition of ACE and/or ECE has a significant application in thetreatment of hypertension and the prevention and treatment ofatherosclerosis.

Inhibition of APN in conjunction with NEP has significant application inthe treatment of pain.

Inhibition of related membrane metallopeptidases has therapeutic effectsin the treatment of tumors, namely ovarian, colorectal, brain, lung,pancreas, gastric and melanoma cancers, and reducing the incidence ofmetastasis, atherosclerosis and/or hypertension. Inhibitions of relatedmembrane metallopeptidases has also therapeutic effects in paincontrolling. Such antinociceptive effects on acute pain are analgesiceffects but also effects on chronic inflammatory pain such as arthritisor inflammatory bowel disease.

Furthermore, inhibition of bacterial or viral metallopeptidase isexpected to have anti-infection effects.

Metallopeptidases playing an important role in pathogen host tissueinvasion and immunological and inflammatory processes, for example thoseof Streptococcus pyogenes, Pseudomonas aeruginosa, Porphyromonasgingivalis and Legionella pneumophila.

Furthermore, bacterial metallopeptidases, especiallyzinc-metallopeptidases play an important role in the diseases caused byproteolytic toxins, such as the toxins of B. anthracis (Anthrax Lethalfactor) and the neurotoxins of C. tetanum and botulinum.

Other metallopeptidases play an important role in various infectionssuch as infections caused by HIV (FR 2 707 169).

The importance of proteinase inhibitors for the treatment of bacterialor viral diseases may be found in J. Potempa, J. Travis, (Proteinases asvirulence factors in bacterial diseases and as potential targets fortherapeutic interaction with proteinase inhibitors. In proteases astargets for therapy. 99, 159–188—Eds K. Helm, B. D. Korant and J. C.Cheronis—Spinger Handbook Exp. Pharm. 140).

The different roles of metallopeptidases are disclosed in Turner et al,(2001) Bioessays, 23, 261–9; Kenny et al, (1977) Proteinases inmammalian cells and tissues); Kenny et al, (1987) Mammalian ectoenzymes;Beaumont et al, (1996) zinc metallopeptidases in health and disease,105–129).

A first subject-matter of the invention is thus the therapeutic use of aSMR1-peptide or a pharmaceutically active amount of said SMR1-peptide,for the preparation of a therapeutic composition for preventing ortreating diseases wherein a modulation of the activity of a membranemetallopeptidase, notably a membrane zinc metallopeptidase, is sought,in a mammal, specifically in a human.

Another object matter of the invention is the therapeutic use of anagent such as a biologically active derivative of SMR1-peptide formodulating the interaction between the endogenous SMR1-peptide and saidmembrane metallopeptidase. Said modulation is a kinetical and/ormolecular one.

“Endogenous” refers to a molecule (herein a SMR1-peptide) that isnaturally expressed or matured in tissues of a patient to be treated.

The invention further relates to the use of an agent that modulates theinteraction between endogenous SMR1 protein or peptide and a membranemetallopeptidase for the preparation of a therapeutic composition forpreventing or treating diseases wherein a modulation of the activity ofsaid membrane metallopeptidase is sought.

The present invention concerns more specifically the therapeutic use ofthe SMR1-peptide or a pharmaceutically active amount of a SMR1-peptide,for the preparation of a medicament for preventing or treating diseaseswherein modulation of NEP-induced degradation of NEP-sensitive peptidesis sought, in a mammal, specifically in human.

As used in the present specification, SMR1-peptide means the SMR1protein, a peptide generated from SMR1, also called a maturation productof the SMR1 protein, or one of the biologically active derivatives ofsaid protein or said maturation product.

In a preferred embodiment, the SMR1-peptide is a compound of structuralformula (1):X₁QHX₂X₃X₄  (SEQ ID NO: 11)

wherein X₁ denotes a hydrogen atom or X₁ represents an amino acid chainselected from the following: X₁=R or G, X₁=RR, or X₁=PRR, or X₁=GPRR, orX₁=RGPRR, or X₁=VRGPRR, X₂ denotes N, G or D, X₃ denotes P or L and X₄denotes R or T.

Preferred peptides comprise peptides of sequence:

QHNPR (SEQ ID NO: 2), RQHNPR (SEQ ID NO: 9) and VRGPRRQHNPR (SEQ ID NO:4) from Ratus norvegius,

QHNLR (SEQ ID NO: 5) and RQHNLR (SEQ ID NO: 6) from Ratus ratus,

GQHGPR (SEQ ID NO: 7) and GQHDPT (SEQ ID NO: 8) from mouse.

In the above amino acid sequences:

Q represents Glutamine,

H represents Histidine,

N represents Asparagine,

G represents Glycine,

P represents Proline,

R represents Arginine,

L represents Leucine,

T represents Threonine,

D represents Aspartic acid, and

V represents valine.

“Biologically active derivatives” of the SMR1-peptide refer tofunction-conservative variants, homologous proteins and peptidomimetics,as well as a hormone, an antibody or a synthetic compound, (i.e. eithera peptidic or non peptidic molecule) that preferably retain the bindingspecificity and/or physiological activity of the parent peptide, asdefined below. They preferably show an ability to bind a membranemetallopeptidase, more particularly NEP. Such binding activity may bereadily determined by binding assays, e.g. by labeling the SMR1derivative or by competition assay with a conventional NEP substrate,optionally labeled.

“Function-conservative variants” are those in which a given amino acidresidue in a protein has been changed without altering the overallconformation and function of the polypeptide, including, but not limitedto, replacement of an amino acid with one having similar properties(such as, for example, polarity, hydrogen bonding potential, acidic,basic, hydrophobic, aromatic, and the like). Amino acids with similarproperties are well known in the art. For example, arginine and lysineare hydrophilic-basic amino acids and may be interchangeable. Similarly,isoleucine, a hydrophobic amino acid, may be replaced with leucine orvaline. Such changes are expected to have little or no effect on theapparent molecular weight or isoelectric point of the protein orpolypeptide. Amino acids other than those indicated as conserved maydiffer in a protein or enzyme so that the percent protein or amino acidsequence similarity between any two proteins of similar function mayvary and may be, for example, from 70% to 99% as determined according toan alignment scheme such as by the Cluster Method, wherein similarity isbased on the MEGALIGN algorithm. A “function-conservative variant” alsoincludes a polypeptide or enzyme which has at least 60% amino acididentity as determined by BLAST or FASTA algorithms, preferably at least75%, most preferably at least 85%, and even more preferably at least90%, and which has the same or substantially similar properties orfunctions as the native or parent protein or enzyme to which it iscompared.

“Allelic variants” are more particularly encompassed, as described ingreater details below.

As used herein, the term “homologous” in all its grammatical forms andspelling variations refers to the relationship between proteins thatpossess a “common evolutionary origin,” including proteins fromsuperfamilies (e.g., the immunoglobulin superfamily) and homologousproteins from different species (e.g., myosin light chain, etc.) (Reecket al., Cell 50:667, 1987). Such proteins have sequence homology, asreflected by their sequence similarity, whether in terms of percentsimilarity or the presence of specific residues or motifs at conservedpositions.

Accordingly, the term “sequence similarity” in all its grammatical formsrefers to the degree of identity or correspondence between amino acidsequences of proteins that may or may not share a common evolutionaryorigin (see Reeck et al., supra). However, in common usage and in theinstant application, the term “homologous,” when modified with an adverbsuch as “highly,” may refer to sequence similarity and may or may notrelate to a common evolutionary origin.

In a particular embodiment, two amino acid sequences are “substantiallyhomologous” or “substantially similar” when greater than 80% of theamino acids are identical, or greater than about 90% are similar andfunctionally identical. Preferably, the similar or homologous sequencesare identified by alignment using, for example, the GCG (GeneticsComputer Group, Program Manual for the GCG Package, Version 7, Madison,Wis.) pileup program, or any of the programs described above (BLAST,FASTA, etc.).

Natural NEP substrates are mainly the peptide hormones: Enkephalins,Substance P, Bradykinin, Angiotensin II and Atrial Natriuretic Peptidewhich play key role in the control of central and peripheral painperception, inflammatory phenomena, mineral exchange and/or arterialtone.

More particularly, one object of the present invention is the use of theabove described therapeutic peptides as analgesic agents by inhibitingNEP at peripheral, spinal and/or supraspinal levels and therebyincreasing the levels and duration of action of central or peripheralendogenous opioids, including enkephalins.

Another object is the use of the above described peptides asantidiarrheal agents.

Another object is the use of the above described peptides asantihypertensive, natriuretic and diuretic agents by inhibitingendogenous AII formation and substance P, BK and ANP inactivation.

A further object is the use of the above described peptides as an agentfor preventing or treating atherosclerosis.

Another object is the use of the above described peptides as an agentfor the treatment of pain including chronic inflammatory pain, such asarthritis or inflammatory bowel disease.

Another object is the use of the above described peptides as an agentfor controlling immuno-inflammatory responses.

Another object is the use of the above described peptides as an agentfor preventing or treating the processes of malignant cell proliferationand dissemination.

Another object of the present invention is the use of the abovedescribed peptides as a substitute in the treatment of drug abuse,notably morphine drug abuse.

Indeed, studies have suggested that the vulnerability to drug abuse andthe development of reward and drug dependence is at least in part, aresult of pre-existent or induced modifications and/or defect of theendogenous opioid system. In this regard, using SMR1-peptide topotentiate the effects of endogenous enkephalins will reduce the variousside-effects (somatic signs of withdrawal) produced by interruption ofchronic morphine or heroin administration.

Still another object of the invention is the use of the above describedpeptides for treating infections such as bacterial or viral diseases.

For purposes of the invention, the term “mammal” is used in its usualtaxonomic sense and specifically includes humans.

For purposes of the invention, a “peptide” is a molecule comprised of alinear array of amino acid residues connected to each other in thelinear array by peptide bonds. Such linear array may optionally becyclic, i.e., the ends of the linear peptide or the side chains of aminoacids within the peptide may be joined, e.g., by a chemical bond. Suchpeptides according to the invention may include from about three toabout 500 amino acids, and may further include secondary, tertiary orquaternary structures, as well as intermolecular associations with otherpeptides or other non-peptide molecules. Such intermolecularassociations may be through, without limitation, covalent bonding (e.g.,through disulfide linkages), or through chelation, electrostaticinteractions, hydrophobic interactions, hydrogen bonding, ion-dipoleinteractions, dipole—dipole interactions, or any combination of theabove.

Preferred peptides according to the invention comprise an amino acidsequence selected from the group consisting of:Glp-His-Asn-Pro-Arg  [SEQ ID NO. 1, where Xaa=Glp]Gln-His-Asn-Pro-Arg  [SEQ ID NO. 2]Arg-Gln-His-Asn-Pro-Arg  [SEQ ID NO. 3]Val-Arg-Gly-Pro-Arg-Arg-Gln-His-Asn-Pro-Arg  [SEQ ID NO 4]Gln-His-Asn-Leu-Arg  [SEQ ID NO 5]Arg-Gln-His-Asn-Leu-Arg  [SEQ ID NO 6]Gly-Gln-His-Gly-Pro-Arg  [SEQ ID NO 7]Gly-Gln-His-Asp-Pro-Thr  [SEQ ID NO 8]

wherein the sequences are shown in N to C configuration, and wherein Glpis pyroglutamate, Gln is glutamine, His is histidine, Asn is asparagine,Pro is proline, Arg is Arginine, Gly is Glycine, Val is Valine, Leu isLeucine, and Thr is Threonine.

In these peptides, by N-terminal cyclization/decyclization, Glp and Glninterconvert.

In addition, certain preferred peptides according to the inventioncomprise, consist essentially of, or consist of an allelic variant of apeptide shown in any of SEQ ID NO. 1–8. As used herein, an “allelicvariant” is a peptide having from one to two amino acid substitutionsfrom a parent peptide, but retaining the binding specificity and/orphysiological activity of the parent peptide. As used herein, “retainingthe binding specificity of the parent peptide” means being able to bindto a monoclonal or polyclonal antibody that binds to one of the peptidesshown in SEQ ID NOS. 1–8 with an affinity that is at least one-tenth,more preferably at least one-half, and most preferably at least as greatas that of one of the actual peptides shown in SEQ ID NOS. 1–8.Determination of such affinity is preferably conducted under standardcompetitive binding immunoassay conditions (Rougeot et al., (E. J. B.219(3) 765–773). “Retaining the physiological activity of the parentpeptide” means retaining the ability of any one of the peptides shown inSEQ ID NOS. 1–8 to bind and to modulate NEP-activity and so to optimizethe local and systemic nociceptive, inflammatory, pressor, and/or ionhomeostatic responses to stress. Determining whether such activity ismodulated is further described later in this specification. The term“allelic variants” is specifically intended to include any humanfunctional homologs of the peptides set forth in SEQ ID NOS. 1–8 whichdo not have the identical amino acid sequence thereof.

Peptides according to the invention can be conveniently synthesizedusing art recognized techniques (see e.g., Merrifield, J. Am. Chem. Soc.85: 2149–2154).

Also part of the invention are preferred peptidomimetics retaining thebinding specificity and/or physiological activity of the parent peptide,as described above. As used herein, a “peptidomimetic” is an organicmolecule that mimics some properties of peptides, preferably theirbinding specificity and/or physiological activity. Preferredpeptidomimetics are obtained by structural modification of peptidesaccording to the invention, preferably using unnatural amino acids, Damino acid instead of L amino acid, conformational restraints, isostericreplacement, cyclization, or other modifications. Other preferredmodifications include without limitation, those in which one or moreamide bond is replaced by a non-amide bond, and/or one or more aminoacid side chain is replaced by a different chemical moiety, or one ofmore of the N-terminus, the C-terminus or one or more side chain isprotected by a protecting group, and/or double bonds and/or cyclizationand/or stereospecificity is introduced into the amino acid chain toincrease rigidity and/or binding affinity.

Still other preferred modifications include those intended to enhanceresistance to enzymatic degradation, improvement in the bioavailabilityin particular by nervous, intestinal, placental and gonad tissues andmore generally in the pharmacokinetic properties and especiallycomprise:

protecting the NH₂ and COOH hydrophilic groups by esterification (COOH)with lipophilic alcohols or by amidation (COOH) and/or by acetylation(NH₂) or added carboxyalkyl or aromatic hydrophobic chain at the NH₂terminus;

retroinversion or reduction isomers of the CO—NH amide bonds ormethylation (or ketomethylene, methyleneoxy, hydroxyethylene) of theamide functions;

substitution of L amino acids for D amino acids;

dimerisation of amino acid peptide chain.

All of these variations are well known in the art. Thus, given thepeptide sequences disclosed herein, those skilled in the art are enabledto design and produce peptidomimetics having binding characteristicssimilar to or superior to such peptides (see e.g., Horwell et al.,Bioorg. Med. Chem. 4: 1573 (1996); Liskamp et al., Recl. Trav. Chim.Pays-Bas 1: 113 (1994); Gante et al., Angew. Chem. Int. Ed. Engl. 33:1699 (1994); Seebach et al., Helv. Chim. Acta 79: 913 (1996)).

The peptides used according to the present invention may be prepared ina conventional manner by peptide synthesis in liquid or solid phase bysuccessive couplings of the different amino acid residues to beincorporated (from the N-terminal end to the C-terminal end in liquidphase, or from the C-terminal end to the N-terminal end in solid phase)wherein the N-terminal ends and the reactive side chains are previouslyblocked by conventional groups.

For solid phase synthesis, the technique described by Merrifield may beused in particular. Alternatively, the technique described by Houbenweylin 1974 may also be used.

For more details, reference may be made to WO 98/37 100.

The peptides used in the therapeutic method according to the presentinvention may also be obtained using genetic engineering methods. Thenucleic acid sequence of the cDNA encoding the complete 146 amino acidSMR1 protein has been described in the PCT Patent Application No. WO90/03891 (Rougeon et al.) For the biologically active peptidederivatives of the SMR1-peptide, for example a derivative of X₁QHX₂X₃X₄(SEQ ID NO: 11), a person skilled in the art will refer to the generalliterature to determine which appropriate codons may be used tosynthesize the desired peptide.

The methods that allow a person skilled in the art to select and purifythe biologically active derivatives that bind to the same targets andhave an agonist or an antagonist biological activity of the SMR1-peptideof the invention are described hereunder.

The biologically active derivative of the SMR1-peptide may be a protein,a peptide, a hormone, an antibody or a synthetic compound which iseither a peptide or a non peptidic molecule, such as any compound thatcan be synthesized by the conventional methods of organic chemistry.

Selection of the biologically active derivatives of the SMR1-peptide ofthe invention is performed both in assessing the binding of a candidateligand molecule to the NEP binding site for the QHNPR (SEQ ID NO: 2)pentapeptide, and in determining the metabolic changes induced by thiscandidate molecule on its target, such as the synthesis and/or releaseof the primary or secondary messenger metabolites as a result of atransduction signal via the protein kinases or adenylate cyclase and theactivation of a protein of the G family or the variation of theenzymatic activity of NEP, specifically on the metabolism of natural NEPsubstrates.

Binding assays of the candidate molecule are generally performed at 4°C. to 25° C. or 37° C. In order to facilitate the reading of thehereinafter described protocol, QHNPR (SEQ ID NO: 2) pentapeptide isalso used instead of or in competition with a biologically activederivative candidate molecule.

Accordingly, another object of the present invention is a process forscreening ligand molecules that specifically bind to the NEP bindingsite for the QHNPR (SEQ ID NO: 2) pentapeptide, comprising the steps of:

a) preparing a cell culture or preparing an organ specimen or a tissuesample (cryosections or slices or membrane preparations or crudehomogenates) containing NEP binding sites for the QHNPR (SEQ ID NO: 2)pentapeptide;

b) adding the candidate molecule to be tested in competition withhalf-saturating concentration of labeled pentapeptide;

c) incubating the cell culture, organ specimen or tissue sample of stepa) in the presence of the candidate molecule during a time sufficientand under conditions for the specific binding to take place;

d) quantifying the label specifically bound to the cell culture, organspecimen or tissue sample in the presence of various concentrations ofcandidate molecule (preferably 10⁻¹⁰ to 10⁻⁵ M).

In said above process, a half-saturating concentration is theconcentration of the labeled QHNPR (SEQ ID NO: 2) pentapeptide whichbinds 50% of the NEP binding sites.

This process also allows to define the relative affinity of thecandidate molecule compared to the QHNPR (SEQ ID NO: 2) affinity.

Another object of the present invention is a process for determining therelative affinity of ligand molecules that specifically bind to the NEPbinding sites for the QHNPR (SEQ ID NO: 2) pentapeptide comprising thesteps a), b), c) and d) of the above process for each candidate moleculeand further comprising the step e) of comparing the affinity of eachcandidate molecule quantified in step d) to the one of the othercandidate molecules.

Another object of the present invention is a process for determining theaffinity of ligand molecules that specifically bind to the NEP bindingsite for the QHNPR (SEQ ID NO: 2) pentapeptide, comprising the steps of:

a) preparing a cell culture or preparing an organ specimen or a tissuesample (cryosections or slices or membrane preparations or crudehomogenates) containing NEP binding sites for the QHNPR (SEQ ID NO: 2)pentapeptide;

b) adding the candidate molecule which has previously been labeled witha radioactive or a nonradioactive label;

c) incubating the cell culture, organ specimen or tissue sample of stepa) in the presence of the labeled candidate molecule during a timesufficient and under conditions for the specific binding to take place;and

d) quantifying the label specifically bound to the cell culture, organspecimen or tissue sample in the presence of various concentrations ofthe labeled candidate molecule (preferably 10⁻¹⁰ to 10⁻⁵ M).

The candidate ligand molecule may be radioactively labeled (³²P, ³⁵S,³H, ¹²⁵I etc.) or nonradioactively labeled (biotin, digoxigenin,fluorescein etc.)

Thus, the present invention also pertains to a process for screeningligand molecules that possess an agonist biological activity on the NEPbinding site of the QHNPR (SEQ ID NO: 2) pentapeptide, comprising thesteps of:

a) preparing a cell culture or preparing an organ specimen or a tissuesample (cryosections or slices or membrane preparations or crudehomogenates) containing NEP binding sites for the QHNPR (SEQ ID NO: 2)pentapeptide;

b) incubating the cell culture, organ specimen or tissue sample of stepa) at concentrations allowing measurement of NEP enzymatic activityunder initial velocity conditions as defined by the method of Example 1(Material and methods) in the presence of the candidate molecule(preferably 10⁻¹⁰−10⁻⁵ M), a half-saturating concentration of QHNPR (SEQID NO: 2) and a NEP substrate during a time sufficient for thehydrolysis of the NEP substrate to take place under initial velocityconditions;

c) quantifying the activity of the NEP present in the biologicalmaterial of step a) by measuring the levels of NEP substrate hydrolysis,respectively in the presence or in the absence of the candidate ligandmolecule and in the presence or in the absence of QHNPR (SEQ ID NO: 2).

In said above process, a half-saturating concentration is theconcentration of the QHNPR (SEQ ID NO: 2) pentapeptide which reduces byhalf the degradation of the NEP substrate.

Another object of the present invention comprises a process forscreening ligand molecules that possess an antagonist biologicalactivity on the NEP binding site of the QHNPR (SEQ ID NO: 2)pentapeptide, comprising the steps of:

a) preparing a cell culture or preparing an organ specimen or a tissuesample (cryosections or slices or membrane preparations or crudehomogenates) containing NEP binding sites for the QHNPR (SEQ ID NO: 2)pentapeptide;

b) incubating the cell culture, organ specimen or tissue sample of stepa) at concentration allowing measurement of NEP enzymatic activity underinitial velocity conditions in the presence of a submaximalconcentration of the XQHNPR (SEQ ID NO: 10) peptide, specifically theQHNPR (SEQ ID NO: 2) peptide and a NEP substrate, in the presence of thecandidate molecule during a time sufficient for the hydrolysis of theNEP substrate to take place under initial velocity conditions;

c) quantifying the activity of the NEP present in the biologicalmaterial of step a) by measuring the levels of NEP substrate hydrolysis,respectively in the presence or in the absence of the candidate ligandmolecule and in the presence or in the absence of QHNPR (SEQ ID NO: 2).

In a preferred embodiment of said above process, a submaximalconcentration is a concentration of pentapeptide which reduces by atleast 50% and preferably by at least 75% the degradation of thesubstrate.

As mentioned above, another metabolic assay in order to assess theagonist or the antagonist activity of the candidate ligand moleculecomprises the incubation of the ligand candidate in the presence of aprimary cell culture or established cell line or tissue sample of rat,mouse or human origins and an endogenous or exogenous NEP substrate anddetermining, either or both quantitatively and qualitatively, thehydrolysis of the NEP substrate.

A preferred tissue sample that is used in the screening methodsaccording to the present invention is a membrane preparation or slicesof spinal cord from rats, a tissue known to be appropriated for NEPactivity measurement.

Other preferred tissue samples that can be used in the screening methodsaccording to the present invention are all peripheral tissuepreparations that are known to be enriched in NEP-peptidase and/or to betargets for SMR1-peptide, for example rat renal outer medulla, placenta,testis, prostate and bone and dental tissues. In addition, such aprocedure can also be applied to tissues and/or cells of mammals (e.g.mouse) and especially human origin or cell lines transfected with humanNEP cDNA, for example MDCK, HEK or COS cells first transfected withhuman NEP cDNA.

Preferred biologically active derivatives of SMR1-peptide and speciallyof X₁QHX₂X₃X₄ (SEQ ID NO: 11) of the therapeutic composition accordingto the present invention have better pharmacodynamic properties than theendogenous natural or synthetic X₁QHX₂X₃X₄ (SEQ ID NO: 11) peptide, andthus possess a longer in vivo half-life as compared to their naturalcounterparts and a better bioavailability in a given tissue/space,especially in nervous, intestine, placental and gonad tissues.

The above-described biologically active derivatives, are also an objectof the present invention.

Thus, the invention also relates to the SMR1 maturation products and thebiologically active derivatives of the SMR1 protein or of its maturationproducts that can be selected according to the screening processeshereinbefore described, provided that they have not the structure offormula (1) above. Indeed, also excluded, is the 146 amino acid proteinconstituting the SMR1 protein itself (PCT Patent Application No WO90/03981). However, the therapeutic use of these molecules that areexcluded as such of the present invention, is a main object of theinstant invention.

Another object of the present invention is a biologically activederivative of the SMR1-peptide characterized by its capacity either toincrease or decrease a metallopeptidase activity or to prevent thenormal interaction between the SMR1-peptide and said metallopeptidase.Preferably, said metallopeptidase is a membrane-zinc metallopeptidase.More preferably, said membrane-zinc metallopeptidase is NEP.

The biologically active derivatives of SMR1-peptide so characterizedalso include SMR1 protein maturation products, provided that they do nothave the structure of formula (1) above.

The SMR1 protein or its maturation products and the biologically activederivatives of the SMR1 protein or of its maturation products used inthe therapeutic compositions according to the present invention havebeen, in a preferred embodiment, selected firstly according to theirability to bind to the same targets as the X₁QHX₂X₃X₄ (SEQ ID NO: 11),specifically QHNPR (SEQ ID NO: 2) peptide, and secondly by theircapacity to modulate hydrolysis of substrate of a metallopeptidase forexample the NEP in vitro or in vivo.

By “modulate”, it is understood that said SMR1-peptide has the capacityeither to increase or decrease (inhibit) the metallopeptidase activityor to prevent the normal interaction between the SMR1-peptide and thesaid metallopeptidase.

The present invention also deals with the use of therapeuticcompositions comprising an effective amount of the SMR1-peptide.

In the methods according to the invention, the peptides orpeptidomimetics according to the invention may be administered by any ofa variety of means. In certain preferred embodiments, administration maybe parenteral, most preferably intravenous. In other preferredembodiments, administration may be intranasal, oral, sublingual,transmucosal, intrarespiratory, or through an inert or iontophoreticpatch.

Dosages of the peptide or peptidomimetic to be administered will dependon the particular patient, the condition, and the route ofadministration, and can be determined empirically by the reduction orelimination linked to the pathological disorders listed above inresponse to an elevating dosage regimen. Preferred dosages are fromabout 0.1 μg/kg to about 1 mg/kg, more preferably from about 1 μg/kg toabout 100 μg/kg, and most preferably from about 1 μg/kg to about 50μg/kg.

In certain preferred embodiments, the peptide or peptidomimeticaccording to the invention is administered together with a secondpharmaceutical, wherein the second pharmaceutical agent is present in anamount insufficient to reduce or eliminate symptoms of the disorder ordisease to be treated, and wherein the peptide or peptidomimeticaccording to the invention and the second pharmaceutical agent actsynergistically to reduce or eliminate symptoms of the disorder ordisease to be treated. Such second pharmaceutical agent may or may notact as a modulator of the metallopeptidase.

“Synergistically” means that the peptide or the peptidomimetic and thesecond pharmaceutical agent together are more effective in reducing oreliminating symptoms of a disorder or disease than either one alonewould be at the same concentration.

The present invention also relates to a molecular complex comprising:

the NEP receptor or the SMR1-binding site of the NEP receptor;

the SMR1-peptide.

The present invention is illustrated in details in the followingexamples without being in any way limited in scope to these specificembodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1-A: Influence of spinal cord membrane protein concentration onSubstance P hydrolysis (25 nM) in the presence or absence of thesynthetic NEP inhibitor, Phosphoramidon, 10 μM. Each point representsthe percent of 3H-substance P hydrolyzed by spinal cord membraneincubated 15 min. at 30° C. in a 250 μl final volume of Tris/HCl buffer.

FIG. 1-B: Time course of Substance P hydrolysis (12.5 nM) by rat spinalcord membrane preparations in the presence or absence of differentpeptidase inhibitors at 10 μM final concentration:—an ACE inhibitor,captopril,—the CPB and DPPIV inhibitors, GEMSA and DPPIV inhibitor. Eachpoint represents the percent of 3H-substance P hydrolyzed by 250 μgmembrane proteins incubated at 25° C. in a 250 μl final volume ofTris/HCl buffer.

FIGS. 2A and B: Met-enkephalinase activity in spinal cord slices, in thepresence or absence of different peptidase inhibitors at 10 μM finalconcentration:—a NEP inhibitor, Phosphoramidon,—a NEP inhibitor,Thiorphan,—the CPB and DPPIV inhibitors, GEMSA and DPPIV inhibitor,—theSMR1-QHNPR (SEQ ID NO: 2) alone or combined with CPB and DPPIVinhibitors. Control represents the Met-enkephalin recovery in theabsence of tissue slice.

FIG. 2-A: Values represent the concentration of intact andimmunoreactive Met-enkephalin (mean of 2 determinations) determined byRIA analysis (μM) and recovered after 20 min. incubation at 25° C. with1 mg fresh tissue slices in a 1 ml final volume of KRBG buffer.

FIG. 2-B: Values represent the quantity of intact Met-enkephalin (meanof 2 determinations) determined by RP-HPLC analysis (peak height at 18.9min. Retention time) recovered after 20 min. incubation at 25° C. with 1mg fresh tissue slices in a 1 ml final volume of KRBG buffer.

FIG. 3-A: Substance P hydrolysis (25 nM) by rat spinal cord slices, inthe presence or absence of different peptidase inhibitors at 10 μM finalconcentration:—a NEP inhibitor, Phosphoramidon,—a NEP inhibitor,Thiorphan,—the CPB and DPPIV inhibitors, GEMSA and DPPIV inhibitor,—theSMR1-QHNPR (SEQ ID NO: 2) alone or combined with CPB and DPPIVinhibitors. Control represents the 3H-substance P hydrolysis in absenceof tissue slice. Each point represents the percent of 3H substance Phydrolyzed by 1 mg fresh tissue slices incubated at 25° C. in a 1 mlfinal volume of KRBG buffer.

FIG. 3-B: Concentration-dependent inhibition by SMR1 QHNPR (SEQ ID NO:2) of 3H-Substance P (12.5 nM) catabolism by rat spinal cord membranepreparations. Comparison with—a NEP inhibitor, Phosphoramidon and,—CPBand DPPIV inhibitors, GEMSA+DPPIV inhibitor. Comparison between theinhibitory activity exerted by QHNPR (SEQ ID NO: 2) peptide alone or incombination with CPB and DPPIV inhibitors. Each point represents themean recovery (in percentages) of intact 3H-substance P after 10 min;incubation at 25° C. with 250 μg membrane protein in 250 μl Tris/HClbuffer (mean of 2 determinations).

FIGS. 4 A and B: Substance P-catalytic activity of various peripheralrat tissue membranes and dose response inhibitory potency of SMR1-QHNPR(SEQ ID NO: 2) (sialorphin) on renal membranes.

FIG. 4-A: Endopeptidase activity of the tissue membrane preparations wasdetermined using 25 nM [3H] substanceP, in the presence of 10 μMbestatin. The enzymatic specific activity expressed in pM/min/μgmembrane protein is done in the absence and in the presence of 10 μMsialorphin or NEP inhibitors (10 μM phosphoramidon or 1 μM thiorphan).

FIG. 4-B: Concentration-dependent inhibition by sialorphin of [3H]substanceP catabolism by rat renal membrane preparations. Each pointrepresents the percentage of 320 nM intact [3H] substanceP recovered andcalculated as percentage of velocity without inhibitor—velocity inpresence of inhibitor/velocity without inhibitor, and (*) represents themean±SD of four determinations. Sialorphin concentration is expressed innM and plotted on a log scale in B right. The protein concentration ofmembrane enzyme were defined according to conditions of measurement ofinitial velocity.

FIGS. 5 A, B and C: Representative profiles of untransformed (FIG. 5-A)and double reciprocal (FIG. 5-B) and Dixon (FIG. 5-C) plot analysis ofthe inhibitory activity of sialorphin on substance P-endoproteolyticactivity by rat renal membranes.

The inhibitory potency of sialophin was measured by using substance P assubstrate at concentrations indicated for A and B, and at 14–24 nM(blank triangle) or 56–105 nM (black triangle) for FIG. 5-C.Concentrations of inhibitor for FIG. 5-A and FIG. 5-B were 0 (blackcircles), 1500 (blank circle) and 4500 nM (blank diamond). Each point ofthe untransformed and double reciprocal plots represents the mean of 2independent determinations of duplicate. Experiments were performed at25° C. in 250 μl Tris-HCl buffer (50 mM pH 7.4) under initial velocitymeasurement conditions.

FIGS. 6 A, B and C: Pain responsiveness to a noxious stimulus in ratsfollowing intravenous administration of sialorphin.

The 3-min test session was performed 5 min after intravenous. rat tailvein administration of sialorphin or its vehicle. Naloxone (0.3 mg perkg body weight) was injected subcutaneously 15 min before sialorphinadministration. Time in centre field sector (FIG. 6-C) (central squareof the open-field without pin) and activity (FIGS. 6-A and 6-B) inperipheral squares (overlayed with pins) during the 3-min test session.Each value represents the mean±sem of 8 animals per group. 1=number ofcrossings of peripheral squares; 2=rearing number on peripheral squares;3=number of escapes responses; 4=number of audible vocalisation and5=time displayed in the central area. Control group corresponded to painresponse in vehicle-treated rats. 50 μg/kg sialorphin, 100 μg/kgsialorphin, 100 μg/kg sialorphin plus naloxone 0.3 mg/kg.

EXAMPLES Example 1 Ex vivo, Exploration of the Functional ConsequencesResulting from the Interaction of SMR1-QHNPR (SEQ ID NO: 2) Peptide withNEP

The consequences of the protection of exogenous NEP-sensitive peptidesby SMR1-Pentapeptide, in the extracellular levels of Met-Enkephalin andSubstance P have been assessed using membrane preparations and freshslices of rat nervous tissues.

1. Materials and Methods

1.1. Animals and Tissue Preparations

Sexually mature (from 7 to 9 weeks) male Wistar rats (Iffa Credo), wereused. Up to the day of experiment, the rats were kept under conditionsof constant ambient temperature (24° C.) and of cycled light (on 8 h/off20 h) with distribution of food and water ad libitum. On the day of theexperiment, the animals were sacrificed by cardiac puncture underpentobarbital (Sanofi, 45 mg/kg body weight, i.p.) or ketamine (Imalgene500, Rhone Merieux, 150 mg/kg body weight, i.p.) anesthesia oralternatively by carbon dioxide asphyxia.

Slices of Fresh Tissue

The organs are rapidly removed, dissected on ice, freed of nerve fibersand of adipose tissues and then washed in cold oxygenated glucose- andbicarbonate-containing Krebs Ringer (KRBG) solution, whose compositionis the following: 120 mM NaCl-5 mM KCl-1.2 mM KH₂PO₄-27.5 mM NaHCO₃-2.6mM CaCl₂-0.67 mM MgSO₄-5.9 mM glucose. The slices of tissues areprepared either manually with the aid of a scalpel (1–2 mm thick), ormechanically with the aid of a “Tissue Chopper” (1 mm thick). Slices arethen dispersed into reaction tubes where they are subjected to threesuccessive washes in ice-cold oxygenated KRBG. Thereafter, they are keptat 4° C. in the same buffer supplemented with 10 μM Bestatin (a membraneaminopeptidase, (APN), inhibitor, Roche) and oxygenated under anatmosphere of 95%O2-5% CO₂ until used immediately, as enzyme source.

Membrane Preparations

The organs dissected out and washed in ice-cold KRBG are homogenized at4° C. in 10 volumes (vol./wt.) of 50 mM Tris/HCl buffered at pH 7.2,using a Teflon-glass homogenizer (3×5 sec.). A first centrifugation of 5min. at 1000×g and 4° C. makes it possible to remove the tissular debrisand the nuclei in the pellet. A second centrifugation of the supernatantat 100 000×g and 5° C. concentrates the membrane fraction into thepellet, which will be superficially washed three times with coldTris/HCl buffer and resuspended in fresh buffer using a Konteshomogenizer, aliquoted and stored at −80° C. while waiting to be used asenzyme source, at least until three months.

1.2 Protein Determination

For the determination of the tissue and membrane protein concentrations,the Bio-Rad DC protein assay (Bio-Rad), was used. As with the Lowryassay, the Bio-Rad kit is based on the reaction of sample proteincontent with an alkaline copper tartrate solution and Folin reagent. Theabsorbance is read at 750 nm from 15 min. to 2 h. after the addition ofreagent. The calibration curve is prepared from dilutions of a standardsolution of BSA (Bovine Serum Albumin) from 0.2 to 1.44 mg/ml protein.

1.3. Measurement of the NEP Enzymatic Activity

1.3.1. NEP Source—Substrates and Inhibitors

For the experiments of analysis of the NEP peptidase activity, an exvivo model using incubations of membrane and fresh tissue slicepreparations from nervous tissues that are known to be appropriate forexploring NEP peptidase activity: i.e. the dorsal zone of rat spinalcord, was first developed. The metabolism rate of the NEP-sensitivepeptides was measured using the both NEP substrates involved in thesignaling of the nociceptive response: the neuropeptides Met-enkephalinand Substance P. Native Met-enkephalin (Peninsula, 10 μm) and modifiedtritiated Substance P: [(3,4³H) Pro²-Sar⁹-Met(O₂)¹¹]-Substance P with aspecific radioactivity of 40 Ci/mmol. (NEN, 12.5–25 nM) were used.

The objective was to measure the NEP-specific endoproteolysis of thesesubstrates. For that, in each test, the hydrolysis of substrate both inthe presence and in the absence of specific synthetic inhibitors of NEP(10 μM Phosphoramidon, Roche and/or 1–10 μM Thiorphan, Sigma), and inall cases in the presence of an inhibitor of APN, the Bestatin (10 μM)was analysed. Furthermore, for studying the functional role ofSMR1-QHNPR (SEQ ID NO: 2), the reaction was carried out in the presenceof the SMR1-peptide alone or combined with specific inhibitors ofmembrane peptidases which could inactivate the QHNPR (SEQ ID NO: 2)peptide by cleaving its C-terminal end: an inhibitor of carboxypeptidaseB (GEMSA, 10 μM, Sigma) and an inhibitor of dipeptidylpeptidase IV(DPPIV inhibitor, 10 μM, Roche).

1.3.2. The Enzymatic Activity Assay

Slices of Fresh Tissue

In the first instance, sections of fresh tissue are preincubated in KRBGmedium containing 10 μM bestatin, at 25, 30 or 37° C. in a constantlyshaken water bath and under an atmosphere of 95%O2-5% CO2, in thepresence or in the absence of NEP inhibitor. At the end of thepreincubation period (15 min.), the medium is replaced with fresh mediumcontaining the substrate alone or combined with NEP inhibitor orSMR1-QHNPR (SEQ ID NO: 2) and the incubation is carried out at the sameincubation conditions as the preincubation. At the end of the incubationperiod (from 5 to 30 min.), the medium is transferred to ice-cold tubescontaining hydrochloric acid, such as the final concentration of HClwill be 0.1 N. Samples are kept at −30° C. until the measurement oftheir intact substrate and its metabolites content.

The temperature and time of incubation as well as the concentration ofsubstrate and of tissue enzyme are defined according to the results suchas the NEP hydrolysis activity will be measured under conditions ofinitial velocity.

Membrane Preparations

The membrane preparations are preincubated in 50 mM Tris/HCl buffered atpH 7.2 and containing 10 μM Bestatin, at 25, 30 or 37° C. in constantlyshaken water, in the presence or in the absence of NEP inhibitor. At theend of the preincubation period (10 min), the substrate is added aloneor combined with NEP inhibitor or SMR1-QHNPR (SEQ ID NO: 2) and theincubation is carried out at the same incubation conditions as thepreincubation. At the end of the incubation period, the reaction isstopped by cooling to 4° C. and adding to hydrochloric acid such as thefinal concentration of HCl will be 0.3 N. Samples are kept at −30° C.until the measurement of their intact substrate and its metabolitescontent.

The temperature and the time of the incubation as well as theconcentration of substrate and of membrane enzyme are defined accordingto the results such as the NEP hydrolysis activity will be measuredunder conditions of initial velocity.

1.3.3. The Detection of the Substrate and its Metabolites

To separate, detect and quantify the intact substrate and itsmetabolites, various techniques (depending on whether the substrate wasradiolabeled or not), were used: two are based on the principle ofreverse-phase chromatography for the selective isolation of the productsof the reaction (C-18 Sep-Pak cartridges and RP-HPLC) and the third isbased on the specific detection of the substrate by radio-immunoassay(RIA).

The C-18 Sep-Pak Cartridges

The C-18 Sep-Pak cartridges (Waters) were used to analyze the hydrolysisof the radiolabeled peptides: they separate compounds according to theirdifferences in polarity. This solid-phase extraction procedure allowsisolating the substrate from its metabolites, since the hydrophobiccharacter of the peptide metabolites is reduced or even lost compared tothe intact peptide substrate.

3H-Metabolites of radiolabeled substance P are eluted in two steps: onewith H₂O—0.1% TFA and the second one with 20% methanol—0.1% TFA, whilethe intact 3H-substance P is eluted in the third step with 70–100%methanol-0.1% TFA. The radioactivity of eluted and isolated compounds isdetermined by liquid scintillation spectrometry.

RP-HPLC (Reverse Phase High Performance Liquid Chromatography)

HPLC is a highly resolutive procedure that allows the isolation anddetection by coupled spectrophotometer analysis, of the non-radioactivepeptides whose concentration is at least 1 to 10 μM. The C-18 RP-HPLC isbased on the same principle as the C-18 Sep-Pak cartridge. Thechromatographic analyses were used to study the hydrolysis ofMet-Enkephalin, that were done on a C-18 LUNA analytical column (150×4.6mm inner diameter, AIT) packed with 5 μm-diameter beads.

RP-HPLC performed with a one-step 30-minute linear gradient ranging fromH₂O—0.1% TFA to 100% acetonitril—0.1% TFA, at a 1 ml/min flow rate,leads to a resolutive separation of the two Met-Enkephalin metabolitesand of the intact substrate. Their identification and relativequantification (peak height) are checked by continuously monitoring theUV absorbance at 254 nm of column outflow.

RIA Assay (Radio-Immuno-Assay)

RIA is a fine analytical method, which allows quantifying compounds,whose concentration is between 1 and 100 nM or even less. Herein, acompetitive RIA system has been used: the quantity of radioactiveantigen bound to the antibody decreases in a manner inverselyproportional to the quantity of antigen contained in the standardsolution or in the sample. The free radioactive antigen is separatedfrom the radioactive antigen—antibody complex by immuno-precipitation.

The activity of enkephalinase NEP is monitored by quantification of thedisappearance of the initial Met-enkephalin substrate. The firstantibody used is a rabbit antibody directed against the C-terminal endof Met-enkephalin (cross-reactivity with metabolites or other peptidesis ≦1%) (Goros et al, J. Neurochem. (1978), 31; 29–39. Radio immunoassayof methionine and leucine enkephalins in regions of rat brain andcomparison with endorphins estimated by a radioreceptor assay). Thesecond antibody is a horse antibody directed against the rabbitimmunoglobulins. The radiolabeled antigen is iodinated Met-enkephalin(¹²⁵I-Met-Enk enkephalin) with a specific radioactivity estimated at3000 Ci/mmol.

Briefly, the Met-enkephalin RIA is performed in 100 mM Tns/HCl bufferedat pH 8.6 and containing 0.1% BSA and 0.1% Triton X 100. Standard (1–100nM) or sample (100 μl), diluted anti-Met-Enkephalin antibody (100 μl,1/2000) and ¹²⁵I-Met-Enk (10000 cpm, 100 μl) are incubated overnight at4° C. Bound and free fractions are separated by immunoprecipitation withthe second anti-rabbit immunoglobulin in presence of polyethylene glycol6000 (6%). After centrifugation, the bound radioactivity of theprecipitate is quantified using a gamma-spectrometer.

2. Results

To specify the inhibitory role of the SMR1-QHNPR (SEQ ID NO: 2) on theNEP enzymatic activity, it was necessary to first develop anexperimental protocol allowing to perform the hydrolysis of Substance Por Met-Enkephalin peptides under conditions of initial velocitymeasurement.

2.1. Search for Experimental Conditions of Initial Velocity Measurementof NEP Endopeptidase Activity

2.1.1. Hydrolysis of Native Met-Enkephalin

In first series of experiment, the slices and the membrane preparationsof spinal cord tissues were incubated at 30° C. in a 1 ml final volumeof KRBG, and at 37° C. in a 0.25 ml final volume of Tris/HCl 50 mM, pH7.2, respectively.

RP-HPLC Analysis

The calibration of the RP-HPLC chromatographic system reveals thatmarker Met-enkephalin is eluted at a retention time of 18.8 min. In thecase of the samples, a peak is identified whose height increasesconsiderably in the presence of a NEP-specific inhibitor: this peak,whose retention time is 18.8±0.2 min., corresponds to the intactMet-enkephalin substrate. Conversely, two peaks having retention timesof 5.8±0.2 min. and 12.8±0.1 min., corresponding to the metabolitesTyr-Gly-Gly and Phe-Met respectively, appear in the absence ofNEP-inhibitors. This result indicates that spinal tissue enzyme hascleaved the substrate predominantly at the Gly³-Phe⁴ amide bond of thepeptide, which already corresponds to enkephalinase activity.

At the level of membrane preparations as well as of fresh tissue slices,a high NEP-specific hydrolysis of the exogenous Met-enkephalin isobserved during the 10 min. incubation at 37° C.: the spinal cordenkephalinase activity provokes a disappearance of the Met-enkephalinpeak and that is reversed in the presence of 10 μM Phosphoramidon or 1μM Thiorphan (80–90% inhibition). In addition, under these conditions,both specific NEP inhibitors ensure the almost complete inhibition ofenkephalinase activity over the time of incubation at 37° C., from 10 to30 min.

Since, the maximum hydrolysis was undoubtedly reached, at 37° C.temperature within the 10 min. incubation, in the next experiments theincubation temperature has been subsequently reduced to 30° C. then to25° C. Effectively, for the fresh tissue slices incubated at 30° C., thelevel of hydrolysis of Met-enkephalin increases with time (from 0 to 30min.). In the same manner, for the membrane preparations incubated at30° C., it is also possible to note an increase in the level ofhydrolysis in relation to the enzyme concentration (from 0 to 2 mg/ml).However, no clear linear relationship could be established.

Indeed, the HPLC chromatography coupled to spectrophotometer analysis isa semi-quantitative technique and the single measurement of the heightsor areas of peaks is not sufficiently precise to calculate quantitativeproportional relationships. Then, to precisely quantify theMet-enkephalin, a specific quantitative RIA detection was used.

2.1.2. Hydrolysis of Modified Tritiated Substance P

The experimental parameters which allow to study, under conditions ofinitial velocity measurement, the hydrolysis of the substrates,Met-enkephalin and Substance P, by nervous tissue-containing NEP, havebeen established.

In that respect, the influence of the membrane protein concentration ofrat spinal cord (from 0.03 to 1 mg/ml, final concentration) on the levelof the Substance P hydrolysis (25 nM), after 15 min. incubation at 30°C., was first tested. As illustrated in FIG. 1-A, the levels of the3H-Substance P degradation, expressed in percent of initial substrateconcentration, increase proportionally from 2 to 25% in a linearrelated-function to membrane protein concentration. A close correlationof r=0.98, n=7 was found in the absence and, of r=0.99, n=7 in thepresence of 10 μM Phosphoramidon. Furthermore, in the same experimentalcondition, the addition of Phosphoramidon results in a clear reductionof the Substance P degradation (50 to 65% protection of exogenouspeptide).

Similarly, the level of Substance P hydrolysis (12.5 nM) as a functionof the incubation time at 25° C. (5–20 min) was also studied. Themembrane protein concentration chosen was 1 mg/ml. The Substance Pcatabolism by spinal cord membranes increases linearly with the time ofincubation, with a close correlation of r=0.97, n=18 (FIG. 1-B).Captopril, (10 μM) a potent inhibitor of the Angiotensin ConvertingEnzyme (ACE) which also cleaves the Substance P, has no effect on theactivity of the enzyme membrane preparations, as well as, for the potentinhibitors of CPB and DPPIV enzymes (protective compounds of theC-terminal SMR1-QHNPR (SEQ ID NO: 2) potential catabolism).

The conditions of initial velocity measurement of the Substance Phydrolysis by spinal cord tissue containing-NEP therefore appear to beestablished. However, the activity of both NEP inhibitors(Phosphoramidon and Thiorphan), does not appeared to be proportionallystable as a function of the incubation duration. Accordingly, the effectof the SMR1-QHNPR (SEQ ID NO: 2) peptide on the NEP activity will besystematically studied in relation to the time of incubation.

2.1.3. Record

The experimental conditions that allow study, under initial velocitymeasurement, of the Met-enkephalin and Substance P catabolism by spinaltissues ex vivo, are reported in the table hereunder.

Preincubation time  10 min (membrane preparations)  15 min (fresh tissueslices) Incubation times  5 min to 30 min. Temperature  25° C. Finalconcentration of membrane  1 mg/ml or tissue protein (spinal cord)Substrate concentration Substance P: 12.5 nM Met-enkephalin 10 μM (HPLC) 20 nM (RIA) Reaction volume  1 ml (fresh tissue slices) 250 μl(membrane preparation) Technique for separating the Sep-Pak + Liquidscintillation counter Metabolites (3H-Substance P) RP-HPLC and RIA(Met-enkephalin) Buffer Trls.HCI 50 mM, pH 7.2 + BSA  0.1% + Bestatln 10μM (membrane preparations) KRBG + BSA 0.1% + Bestatin 10 μM Oxygenatedunder 95% 02–5% CO₂ (Fresh tissue slices)

2.2 Study of the Functional Consequences Resulting from the Interactionof the SMR1-QHNPR (SEQ ID NO: 2) Peptide with NEP

2.2.1 Degradation of Met-Enkephalin by NEP Spinal Cord

The effect of a fixed concentration of SMR1-QHNPR (SEQ ID NO: 2) (10 μM)on the Met-enkephalinase activity of spinal cord slices underexperimental conditions defined in paragraph 2.1.3, was first tested.

RP-HPLC Analysis

As illustrated in FIG. 2-B, the HPLC analyses show a strong NEP-specifichydrolysis of the Met-enkephalin substrate by spinal cord slices withinthe 20 min. incubation at 25° C. Phosphoramidon at a concentration of 10μM ensures the complete inhibition of Met-enkephalinase activity andaddition of Thiorphan (10 μM) results in a clear reduction by 80% of theMet-enkephalin degradation.

In the same experiment, the QHNPR (SEQ ID NO: 2) peptide, at 10 μMconcentration, alone or combined with the inhibitors of CPB and DPPIVproteases, has an inhibitory activity of 70 or 80%; thus theSMR1-Pentapeptide is able to enter into competition with theenkephalin-pentapeptide for the NEP binding sites, both being in equalconcentrations. As in case of Substance P degradation by spinal membranepreparations, the inhibitors of CPB and DDPIV alone do not have anyintrinsic inhibitory activity on the Met-enkephalin degradation by freshspinal slices. Furthermore, they apparently are no need for protectingSMR1-QHNPR (SEQ ID NO: 2) itself, especially at its C-terminal end, fromthe peptidase activity potentially present in slices of fresh spinaltissue.

In order to finely quantify the NEP activity and inhibition, the sameexperiment has been analyzed with the aid of the specific Met-EnkephalinRIA.

RIA Assay

As a whole, the crude results obtained by the reverse phase-HPLCtechnique are confirmed by those derived from RIA assay (FIG. 2-A).Within the 20 min incubation period at 25° C., the Phosphoramidon,Thiorphan, as well as SMR1-QHNPR (SEQ ID NO: 2) appear as very potentcompounds for protecting Met-enkephalin from NEP degrading activity.Thus, at concentration of 10 μM, they almost totally prevented thedegradation of 10 μM Met-enkephalin by fresh spinal cord tissue: 96%,100% and 96% protection, respectively.

In conclusion, all these results show the negative regulatory roleexerted by the SMR1-QHNPR (SEQ ID NO: 2) peptide on theMet-enkephalinase activity of rat nerve tissues, ex vivo.

2.2.2 Degradation of Substance P by NEP Spinal Cord

SMR1-QHNPR (SEQ ID NO: 2), an Inhibitor of the NEP Activity on SubstanceP Catabolism

In a first instance, the effect of QHNPR (SEQ ID NO: 2) peptide on thehydrolysis of Substance P was searched as it was already done inrelation to Met-enkephalin. For that, spinal cord slices were used and akinetic over a 30-min. incubation period was performed under theconditions of initial velocity measurement defined in 2.1.3.

As illustrated in FIG. 3-A, Substance P hydrolysis reaction effectivelytakes place under initial velocity conditions: a close relationship ofr=0.99 was found between the percentage of Substance P hydrolysis andthe incubation time at 25° C. Ten μM Phosphoramidon or 10 μM Thiorphanexhibits relatively the same inhibitory activity (60–65% inhibition).The QHNPR (SEQ ID NO: 2) peptide (10 μM) is found to be an efficientinhibitor: 75% inhibition of Substance P degradation when it is alone,more than 90% when it is combined with GEMBA (10 μM) and DPPIV inhibitor(10 μM). These latter, however, appear to exhibit an inherent inhibitingactivity of Substance P degradation by fresh spinal tissue.

Otherwise, in this experiment, the effect of inhibitors isproportionally stable as a function of the duration of incubation overthe 30 min. incubation period (r=0.99).

Determination of the IC₅₀

The dose-response curve of the SMR1-QHNPR (SEQ ID NO: 2) inhibitoryeffect on 3H-Substance P degradation by spinal cord membranepreparations, shown in FIG. 3-B right panel, allows the calculation ofan IC50 value (concentration of the inhibitor reducing by half thedegradation of 3H-substance P) of about 1.10⁻⁷ M. In the sameexperiment, comparison with Phosphoramidon reveals that protection ofthe exogenous Substance P by SMR1-QHNPR (SEQ ID NO: 2) is stillequivalent t0 that obtained with Phosphoramidon (FIG. 3-B left panel).Furthermore, the QHNPR (SEQ ID NO: 2) peptide combined with theinhibitors of CPB and DPPIV exhibits a very high NEP inhibitingactivity, greater than that of phosphoramidon (FIG. 3-B, left panel).

2.2.3. Record

The metabolism rate of the NEP-sensitive peptides has been measuredusing tritiated substrate coupled to chromatographic analysis (SubstanceP) or using native substrate coupled to specific RIA quantification(Met-enkephalin). Under conditions of initial velocity measurement ofthe NEP enzymatic activity, an almost complete inhibition of exogenousMet-enkephalin or Substance P catabolism resulting from addition ofSMR1-Pentapeptide has been observed: the concentration of SMR1-QHNPR(SEQ ID NO: 2) which reduces by half the degradation of Substance P byspinal cord tissues, was calculated to be 1.10⁻⁷M and its inhibitorypotency is equivalent to that of two well-known NEP-specific inhibitors,Thiorphan and Phosphoramidon. From these results it appears that, exvivo, the SMR1-Pentapeptide efficiently prevents the spinal NEP-induceddegradation of both neuropeptides involved in the control of spinal painperception, e.g. Substance P and Met-Enkephalin.

Example 2 SMR1-QHNPR (SEQ ID NO: 2) (Sialorphin), an Inhibitor of theSubstance P-Catabolism by Peripheral Tissues

The first results showed the regulatory role exerted by the sialorphinpeptide on the enkephalinase activity of rat nerve tissues. The sameapproach was applied to peripheral tissue membrane preparations that areknown to be enriched in NEP-peptidase and/or to be targets forsialorphin, in vivo, i.e., renal outer medulla, intestine mucosa,placenta, prostate, dental and bone tissues, as well as submandibularepithelium (Rougeot, C. et al, American Journal of Physiology, (1997)273, R1309–20; Sales et al., (1991) Regulatory Peptides 33, 209–22) andreviewed by Kenny et al., (1987), Mammalian ectoenzymes 169–210. Thereis evidence that, almost all these tissues contain substance P releasedfrom peripheral parasympathetic and sensory nerve terminals acting nearthe site of release on target cells that contain the neurokininreceptors to modulate the particular tissue function (McCarson et al.,(1999), Neuroscience 93, 361–70). Thus, it appeared that theneuropeptide could be regarded as a relevant biologically NEP substrateat the periphery. However, substance P is cleaved potently by NEP andACE membrane-bound peptidases, and both enzymes are highly distributedin the renal epithelium (Skidgel et al., (1985), Progress in Clinical&Biological Research 192, 371–8).

The specificity of the peptidase assay was assessed by testing theinhibitory efficacy of selective peptidase inhibitors (at 1–10 μM finalconcentration to induce maximum inhibitory response) on theendoproteolysis of 3H substance P by the various tissue-membraneenzymes, and by analysing the selective formation of the NEP-relatedtritiated product of the reaction that was defined, as above usingspinal tissue. In addition, under standard conditions of initialvelocity measurement, bestatin (10 μM) was added in the incubationmedium to prevent unselectively the membrane aminopeptidase activities.

As shown in FIG. 4-A and in agreement with previous data, male ratkidney contained the highest level of substance-P-hydrolytic specificactivity: 197 pM/min/μg membrane protein from which 61±10%, n=4 was dueto NEP activity and 38±12% was the result of ACE activity. Sialorphininhibited the renal membrane activity with equal effectiveness than thephosphoramidon NEP inhibitor, i.e., 60±5% of maximum inhibitory response(n=9). When inhibitory efficacy was tested on purified rabbit renal NEPby using substance P as substrate (Vi=140 pM/min/μg enzyme), it has beenfound that the substance P catabolism by the soluble-enzyme was alreadyinhibited by sialorphin (46% for 5 μM).

Furthermore, the inhibitory effect of sialorphin on 3H substance-Pdegradation by rat kidney, shown in FIG. 4-B, was strictlydose-dependent (r=0.970, n=20) thus allowing to evaluate the inhibitorypotency with IC50 values in the [0.5–1] micromolar range. Thisinhibitory potency is closely related to that obtained using purifiedrabbit renal NEP and synthetic specific fluorogenic substrate, i.e. 0.6μM or using rat spinal NEP, i.e. 0.4 μM.

All these results indicated that the renal NEP/sialorphin molecularinteraction that has been already evidenced after in vivo tissue uptakemight lead to a physiological action, for instance protection of theNEP-induced metabolism of regulatory peptides present within thistissue, such as substance P, an humoral vasodilatory-proinflammatorymediator and autonomic neurotransmitter. Kidney that contains thehighest NEP activity seems to be also a major site of ANP metabolism(Webb et al., (1989), Journal of Cardiovascular Pharmacology 14,285–93). Thus, one can hypothesise that at renal sites sialorphin couldalso play a role in potentiating the physiological effects of thispeptide messenger whose action is clearly regulated by NEP (Kenny et al.(1988) FEBS Letters 232, 1–8); ANP is a vasodilatory and natriureticfactor mediating physiological regulation of blood pressure, body fluidcirculation and mineral homeostasis.

In rat placenta and prostate tissues, two other peripheral tissuesrichest in NEP, the levels of substanceP-endoproteolytic activity was12–14-fold lower than in kidney and 74±10%, n=5 was shown to be due toNEP while only 8% was the result of ACE. Moreover, sialorphin decreasedsubstance P degradation from these tissues by 70±3%. Prostate tissue wasnot seen to be accessible to a systemically administered hydrophiliccompound such as sialorphin 3H-peptide, however this tissue is alreadyable to synthesise it, suggesting a potential important local role forsialorphin, as inhibitor of endogenous peptidergic signal inactivation,such as substance P (Rougeot et al., (1994), European Journal ofBiochemistry 219, 765–73; Rougeot, C. et al. (1997), American Journal ofPhysiology 273, R1309–20).

The most striking result comes from the observation that the rat innerdental tissue which is one of major targets for sialorphin, in vivo,showed high levels of substance-P endoproteolytic activity, i.e. 44pM/min/μg membrane protein (Rougeot, C. et al. (1997), American Journalof Physiology 273, R1309–20. The addition of NEP inhibitors reduced3H-substance P catabolic process by 53±4%, while ACE inhibitor reducedit by 21% and the sialorphin by 39±14%, n=4. In line with this result,is the present demonstration of sialorphin inhibitory efficacy by75±10%, n=4 on the ectopeptidase-sensitive substance P degradation byinner bone tissues. The possible involvement of sialorphin in NEPfunction within these tissues is supported by the observation that theenzyme's localisation and activity in rat peripheral tissues wellcoincide with the tissue distribution and the density of sequestrationsites for sialorphin; and these tissues included the skeletal andalveolar bones and the periosteal surfaces (Rougeot, C. et al. (1997),American Journal of Physiology 273, R1309–20; Sales et al., (1991),Regulatory Peptides 33, 209–22; Llorens et al., (1981), European Journalof Pharmacology 69, 113–6). Furthermore, from the inner bone and dentalmembrane extracts the major sialorphin-associated molecule exhibit a pIof 5.2±0.4, (n=5) and 5.7±0.6, (n=3), respectively, thus wellcorrelating to the pI for NEP (5.5). However the physiologicalNEP-sensitive effector peptide(s) implicated in the regulation ofskeletal and dental mineralisation and/or resorption processes remain tobe identified.

A similar situation occurs for other structures previously demonstratedto be labelled by NEP-inhibitor or sialorphin such as the rat SMG(Rougeot, C. et al. (1997), American Journal of Physiology 273,R1309–20; Sales et al., (1991), Regulatory Peptides 33, 209–22). Indeed,the SMG level of substance-P-hydrolytic specific activity was found tobe 4.2 pM/min/μg membrane protein and 55±12%, n=4 was due to NEPactivity and 20% to ACE activity, whereas the addition of sialorphinresulted in 79% inhibition. This gland is the major site of sialorphinsynthesis where it might be thus involved in the regulation of localprotein and/or fluid secretions through modulation of activity ofsubstance P, an extremely potent sialolog compound in rat (Yu et al.,(1983), Experimental Biology &Medicine 173, 467–70).

Moreover some other richly supplied area is the gut, especially theintestinal wall which expresses NEP, contains substance P extrinsicsensory and enteric neurons and sialorphin uptake sites (Rougeot, C. etal. (1997), American Journal of Physiology 273, R1309–20; Holzer et al.,(1997), Pharmacology & Therapeutics 73, 219–63). The substance-Pendoproteolysis by membrane fractions of the rat intestine was found tobe 93.5 pM/min/μg membrane protein. The inhibition profile showed thatin the presence of NEP inhibitors 51% of the exogenous 3H substance Pwas saved from catabolic process, whereas addition of sialorphin inducedpowerful inhibitory response with 87±17%, n=3 protection.

Taken together, these results strongly indicate that in vitro thesialorphin efficiently prevents the endopeptidase-induced degradation ofthe neuropeptide or humoral mediator, substance P, which is availablelocally in a number of tissues where NEP and sialorphin synthesis and/oruptake are also located. This suggests that the circulating sialorphincontribute in vivo to the regulation of peripheral vasodilatory andproinflammatory actions of substance P. Furthermore, as a number ofperipheral effects of circulating ANP are under NEP regulation, One canhypothesise that sialorphin also modulates its vasorelaxant, diureticand natriuretic actions, especially at renal, intestine, bone andsubmandibular sites (Kenny et al., (1988), FEBS Letters 232, 1–8; Vargaset al., (1989), Endocrinology 125, 2527–31; Gonzalez et al., (2000),Peptides 21, 875–87.

Example 3 Sialorphin has Kinetic Behavioural Characteristics of aCompetitive Inhibitor

In order to determine inhibitor modality, all the measures of initialvelocity of the renal enzymatic reaction were plotted versus substrateconcentration for several fixed inhibitor concentrations or versusinhibitor concentration for fixed substrate concentrations.

The pattern of lines in the untransformed (FIG. 5-A) anddouble-reciprocal plot (FIG. 5-B) as well as Dixon plot (FIG. 5-C)analyses of the inhibition by sialorphin on [3H] substance P catabolismby renal membrane are the characteristic signature of a competitiveinhibition. Competitive inhibitors function through binding at theenzyme active site, hence competing directly with the substrate for theactive free enzyme. Hence, the competition between sialorphin andsubstance P has the kinetic effect of raising the apparent Km of theenzyme for substrate by 2–5 fold.

Otherwise, tissue-uptake of the sialorphin peptide involves a complexmolecular species, including a cation mineral element, as the peptidewas only recovered in the presence of a strong divalent metal ionchelating agent (Rougeot, C. et al. (1997), American Journal ofPhysiology 273, R1309–20). Furthermore chelating-FPLC analyses showedthat the sialorphin has a selective and strong zinc-chelating group,likely involving its histidine residue. The zinc ion, an essentialcomponent of the NEP catalytic site, is a common target of syntheticpotent NEP inhibitors described elsewhere. Indeed, they were designedwith a phosphate (phosphoramidon) or thiol (thiorphan) or hydroxamategroups (kelatorphan) as zinc-coordinating moiety, fitting the activesite of metallopeptidase (reviewed by Roques et al., (1993),Pharmacological Reviews 45, 87–146).

Taking the kinetic behaviour of sialorphin into account in addition tothe fact that the in vivo peptide interaction with its membrane receptorsites involved multivalent mineral ion, one can postulate that thesialorphin shares some structural communality with the transition stateof the reaction, thus allowing to optimise interactions with groups inthe enzyme active site, for instance as a zinc coordinating ligand.

The crystal structure determination of NEP when complexed withsialorphin would allow one to gain insight into the distinctive bindingmode of this natural competitive inhibitor.

Example 4 Sialorphin, a New Class of Natural Analgesic

NEP plays a pivotal role in the control of biological activity of theneuropeptide signals involved in conveying sensory information ofdifferent modalities from the peripheral tissues (cutaneous, muscularand visceral areas) to multiple central and peripheral nervous systemneuronal circuits. Prominent among these mediators is substance P, asensory neurotransmitter and enkephalins, the analgesic neuromodulators(Dickenson, (1995), British Journal of Anaesthesia 75, 193–200). It isdemonstrated here below that sialorphin potently prevents theirextracellular catabolism by rat spinal tissues, in vitro.

The importance of enkephalins in modulating nociceptive information hasbeen evidenced in pre-proenkephalin gene-deficient mice, which exhibitedsignificant decrease in nociceptive thresholds (Konig, M. et al. (1996)Nature 383, 535–8). Conversely, using inhibitors of membrane-bound zincmetallopeptidases, NEP and APN which are both involved in the rapidinactivation of the enkephalins, resulted in potent analgesic responses(Chen et al., (1998) Proceedings of the National Academy of Sciences ofthe United States of America 95, 12028–33).

To extend the insight into the in vivo possible antinociceptive propertyof sialorphin through enkephalin-degrading enzyme inhibition, itseffects were assessed in rat model of pain, i.e. the pin pain test,(Hebert et al. (1999) Physiology & Behavior 67, 99–105) in which thevarious behavioural parameters of pain responses were recorded with a3-min cutoff time.

The in vivo activity of sialorphin was tested on the pin pain assayusing male rats (350–400 g, Charles Rivers). The experimental deviceconsists in an open-field (45×45×40 cm) which is divided into nine equalsquares (150×150 mm), eight of them are peripheral and one is central.The peripheral squares are overlayed with stainless steel pins (2/cm2,length 8 mm and diameter 0.6 mm). The test consisted in placing the ratin the central square of the open-field and recording its differentbehaviours (cut-off time, 3 min). Two days before the pain test, therats were familiarised with the experimental device without pins for 20min, so as to reduce the stress linked to the spatial neophobia. Allstatistical analyses were carried out using the Statview 5 statisticalpackage.

As shown in the FIG. 6-A, intravenous-administered sialorphin-treatedrats emitted less vocalisation compared to vehicle-controls anddisplayed locomotor and exploratory activities in the peripheralpin-areas. For instance, 100 μg per kg body weight sialorphin producedprofound analgesic response, as it induced significant increase in thefrequency of crossings peripheral squares during the course of 3-mintrial: 11.13±1.43, n=8 versus control 2.88±0.44, n=8, p≦0.001 by ANOVAand unpaired t-tests, as well as of rearings on peripheral squares:3.88±0.83 versus 0.75±0.41, p≦0.005. In parallel, it induced significantdecrease in the number of audible vocalisation—(0.25±0.16 versus7.25±3.13 p≦0.05) and escape—(0.13±0.13 versus 6.88±2.47, p≦0.05)responses to painful stimuli.

Hence, sialorphin-treated rats displayed powerful morphine-like levelsof analgesia, i.e., 74–97% analgesia at 100 μg/kg given intravenously,in the pin pain tests, in rat.

Furthermore, in a second test trial, the sialorphin-effect on thesebehavioural parameters of noxious response, showed in FIG. 6-B, werereversed by 42–63% by prior administration of 0.3 mg per kg body weightnaloxone (subcutaneous-injection) a μ-opioid receptor antagonist(vocalisation parameter was 20% naloxone-reversible). In addition, asshown in FIG. 6-C, sialorphin-treated rats spent significantly less timein the central area of the open-field that is not pin-overlayed thancontrols (57.75±21.30 sec versus 155.13±14.21 sec, p=0.0019), and thisbehaviour was 56% nalaxone-reversible (112.38±17.44 sec). Thisdemonstrates that μ-opiate receptor is required for completepharmacological sialorphin-induced analgesic effect, thus supportingendogenous opioidergic mediation of sialorphin-induced analgesia.Mu-receptor dependent opioidergic pathways have an essential role inspinal and supraspinal control of nociceptive inputs and inmorphine-induced analgesia (Besse et al., (1990) Brain Research 521,15–22; Matthes et al., (1996), Nature 383, 819–23; Sora, I. et al.,(1997) Proceedings of the National Academy of Sciences of the UnitedStates of America 94, 1544–9). Thus sialorphin might produce a part ofits analgesic effects through potentiation of endogenous μopioid-dependent pathways resulting to spinal and brain antinociception.

Example 5 Study of the Activity of the QHNPR (SEQ ID NO: 2) Peptide inthe Aversive Light Stimulus Avoidance Test 1—Materials and Methods

1.1—Animals

Twenty four male SPF Wistar/AF rats weighing from 300 to 320 g wereused. Upon reception, the rats were weighed, marked and distributed, ingroups of 3, into F-type polycarbonate cages. The animals were housed inan air-conditioned animal house at a temperature of 22–24° C. Food anddrink was available to the rats ad libitum. They were subjected to a12-hour light/dark cycle (light from 8 pm to 8 am).

After a period of familiarization with the laboratory conditions of oneweek, the 24 rats were randomly divided into 2 groups (n=12). The ratsfrom the various groups were all handled in the same way and under thesame conditions.

1.2—Materials

Device for Aversive Light Stimulus Avoidance Conditioning (ALSAT)

The experimental device consists of an isolated cage (50×40×37 cm) whichis brightly lit and comprises two levers: one lever is active, making itpossible, when it is operated, to obtain 30 seconds of darkness,followed by the return of the light, whereas the other lever is inactive(not positively reinforced). Pressing the active lever during the periodof darkness does not produce further periods of darkness. The rat isplaced in the cage for 20 minutes and the number of times each lever ispressed is counted during the experiment.

The test battery, composed of 4 conditioning devices, is entirelyautomated and computer-controlled. Thus, no experimenter is present inthe room during the test.

1.3—Experimental Procedure

This model uses the aversion of the rat to a brightly lit environment.During the familiarization session and the learning session, the ratlearns to control the aversive light environment of the test device inthe context of operant conditioning: the animal learns to press theactive lever in order to obtain periods of darkness.

The learning test is made up over two sessions:

-   -   Session 1, familiarization with the experimental device (day 1);    -   Session 2, learning test (day 2).

Variables Recorded

-   -   The number of times the active lever (AL) is pressed;    -   The number of times the inactive lever (IL) is pressed.

1.4—Products

Products Product QHNPR 0.01 N Distilled peptide acetic acid PBS waterOrigin BACHEM Riedel de Haèn Fluka Chaix and Switzerland GermanySwitzerland Du Marais France Preparation Dissolved in Diluted in methodacetic acid distilled water diluted in distilled water, and bufferedwith D-PBS

1.5—Administration of Products

The QHNPR (SEQ ID NO: 2) peptide is suspended in a proportion of 500 μgper 5 ml of 0.01N acetic acid, and then diluted with PBS in order to beadministered at the dose of 50 μg/kg, via the i.v. route, in the dorsalcaudal vein of the rat, 1 minute before the test.

Product administration protocol Administration before the Rats per DoseVolume test Group group Treatment (μg/kg) Route ml/kg (minutes) Vehicle12 Acetic acid + — I.V. 0.7 1 PBS Peptide 12 FG6-005 50 I.V. 0.7 1

1.6—Statistical Analyses

A two-sided probability unpaired t-test was used to compare thelever-pressing activity of the two groups of rats.

In order to evaluate the distinction between the two levers, a two-sidedprobability paired t-test was used to compare the number of times theactive lever was pressed with the number of times the inactive lever waspressed, within each of the groups.

The results are expressed as mean±standard error of the mean (SEM).

2—Results

2.1—Total Number of Times the Two Levers were Pressed During the TestSessions

During the two test sessions, the rats treated with the QHNPR (SEQ IDNO: 2) peptide proved to be significantly less active than the controlrats in the aversive light stimulus avoidance test.

Total number of times the two levers were pressed during the testsessions

(mean ± SEM) Treatment Vehicle Peptide I.V. 50 μg/kg, I.V. Unpairedt-test (n = 10) (n = 10) (two-sided prob.) Session 1 25.58 ± 6.15 10.08± 1.98 t = 2.40; p < 0.05 Session 2 21.50 ± 5.09  5.25 ± 5.09 t = 3.08;p < 0.01

2.2—Distinction Between the Levers

During the two test sessions, the control rats press the active leversignificantly more than the inactive lever.

This is not the case with the rats treated with the QHNPR (SEQ ID NO: 2)peptide, which make no distinction between the two levers.

Distinction between the levers during the test sessions

(mean ± SEM) Treatment Vehicle Peptide I.V. 50 μg/kg, I.V. (n = 10) (n =10) Session 1 Number of times AL pressed 14.17 ± 3.52 4.83 ± 1.02 Numberof times IL pressed 11.42 ± 2.68 5.25 ± 1.14 Paired t-test (two-sidedprob.) t = 2.30; p < 0.05 T = 0.49; N.S. AL vs IL Session 2 Number oftimes AL pressed 12.83 ± 3.22 2.67 ± 0.47 Number of times IL pressed 8.67 ± 1.97 2.58 ± 0.94 Paired t-test (two-sided prob.) t = 2.63; p <0.05 T = 0.13; N.S. AL vs IL AL: active lever; IL: inactive lever.

3—CONCLUSION

Under these experimental conditions, during the two test sessions, therats treated with the QHNPR (SEQ ID NO: 2) peptide prove to besignificantly less active than the control rats in the aversive lightstimulus avoidance test. Furthermore, they show no learning, since theymake no distinction between the two levers.

Either these rats are less sensitive to the nociceptive light stimulus,or they are more sensitive to the stress of the experimental lightenvironment. Given that it has been directly observed that these ratshave satisfactory locomotor and exploratory activity during the tests,it is the fact that they are less sensitive to the aversive stimuluswhich explains their performance. The peptide thus exhibits analgesicactivity.The control rats show satisfactory activity with regard to manipulatingthe levers and make a distinction between the active lever and theinactive lever, both during the first session and during the secondsession.

1. A method for treating or reducing the severity of a disease ordisorder mediated by a membrane metalloproteinase comprising:administering to a subject in need thereof an amount of a SMR-1(submandibular rat 1 protein) peptide sufficient to modulate theactivity of a metalloproteinase, wherein the SMR1-peptide comprises asequence shown by formula (1):X₁QHX₂X₃X₄  (SEQ ID NO: 11) wherein X₁ is absent from formula (1) or isselected from the group consisting of R, G, RR, PRR, GPRR, RGPRR andVRGPRR, X₂ is N, G or D, X₃ is P or L and X₄ is R or T.
 2. The method ofclaim 1, wherein said metalloproteinase is a membrane-zincmetallopeptidase.
 3. The method of claim 1, wherein an amount of saidSMR-1 peptide sufficient to modulate NEP (neutral endopeptidase)-induceddegradation of an NEP sensitive peptide is administered to a mammal. 4.The method of claim 3, wherein said amount of SMR-1 peptide issufficient to inhibit NEP.
 5. The method of claim 3, wherein said amountof SMR-1 peptide is sufficient to inhibit enkephalin degradation.
 6. Themethod of claim 1, wherein said amount of SMR-1 peptide is sufficient toprovide an antinociceptive effect.
 7. The method of claim 1, whereinsaid amount of SMR-1 peptide is sufficient to provide analgesia.
 8. Themethod of claim 1, wherein said disease or disorder is characterized bychronic inflammatory pain.
 9. The method of claim 8, wherein saiddisease or disorder is arthritis or inflammatory bowel disease.
 10. Themethod of claim 1, wherein said disease or disorder is diarrhea.
 11. Themethod of claim 1, wherein said amount of the SMR-1 peptide issufficient to inhibit endogenous A II formation and inhibit inactivationof substance P, BK (bradykinin) and/or ANP (atrial natriuretic peptide).12. The method of claim 1, wherein said disease or disorder ishypertension.
 13. The method of claim 1, wherein said amount of SMR-1peptide is sufficient to exert natriuretic activity.
 14. The method ofclaim 1, wherein said amount of SMR-1 peptide is sufficient to exert adiuretic activity.
 15. The method of claim 1, wherein said disease ordisorder is atherosclerosis.
 16. The method of claim 1, wherein saiddisease or disorder is characterized by malignant cell proliferationand/or dissemination.
 17. The method of claim 1, wherein said disease ordisorder is a viral or bacterial infection.
 18. The method of claim 1,wherein said disease or disorder is characterized by an abnormalimmuno-inflammatory response.
 19. The method of claim 1, wherein saiddisease or disorder is drug abuse.
 20. The method of claim 1, whereinthe SMR1-peptide is the SMR1 protein, or a maturation product of theSMR1 protein.
 21. The method of claim 1, wherein the SMR1-peptidecomprises one or more amino acids in the D-form.
 22. The method of claim1, wherein the SMR1-peptide further comprises a group for enhancingbioavailability or enhancing resistance to enzymatic degradation. 23.The method of claim 1, wherein the SMR1-peptide is associated with asecond pharmaceutical agent that acts synergistically with theSMR1-peptide.
 24. The method of claim 1, wherein X₁ is absent fromformula (I).
 25. The method of claim 1, wherein X₁ is R or G.
 26. Themethod of claim 1, wherein X₁ is RR, PRR, or GPRR.
 27. The method ofclaim 1, wherein X₁ is RGPRR or VRGPRR.
 28. The method of claim 1,wherein X₂ is N.
 29. The method of claim 1, wherein X₂ is G.
 30. Themethod of claim 1, wherein X₂ is D.
 31. The method of claim 1, whereinX₃ is P.
 32. The method of claim 1, wherein X₃ is L.
 33. The method ofclaim 1, wherein X₄ is R.
 34. The method of claim 1, wherein X₄ is T.35. A method for treating or reducing the severity of a disease ordisorder mediated by a membrane metalloproteinase comprising:administering to a subject in need thereof an amount of a modified SMR-1(submandibular rat 1 protein) peptide sufficient to modulate theactivity of a metalloproteinase, wherein the modified SMR1-peptidecomprises a sequence shown by formula (1): X₁QHX₂X₃X₄ (SEQ ID NO: 11),wherein X₁ is absent from formula (1) or is selected from the groupconsisting of R, G, RR, PRR, GPRR, RGPRR and VRGPRR, X₂ is N, G or D, X₃is P or L and X₄ is R or T; wherein said modification is at least oneselected from the group consisting of incorporating of one or moreD-amino acids into the SMR1 peptide sequence, protecting one or moreamino acid side chains with a protecting group, protecting one or moreNH₂ or COOH functions in the SMR1 peptide, replacing of one or moreamide bonds in the SMR1 peptide with a non-amide bond, introducing oneor more double bonds into the SMR1 peptide sequence, introducingretroinversion or reduction isomers of the CO—NH amide bonds,methylation of at least one amide function in the SMR1 peptide,modifying the SMR1 peptide to have more than one N or C terminus,introducing one or more conformational restraints into the SMR1 peptide,cyclizing the SMR1 peptide, introducing stereospecificity into the SMR1peptide, and dimerizing the SMR peptide.